1. Field of the Invention
The present invention relates to a sensor device for label-free detecting nucleic acid sequences, comprising an optical whispering gallery mode (WGM) resonator. Furthermore, the present invention relates to a sensing method for label-free detecting nucleic acid sequences using the WGM resonator. Applications of the invention are available e.g. in the fields of research and clinical nucleic acid sequence detection or screening.
2. Brief Description of the Related Art
It is generally known that specific detection of nucleic acids, like DNA and RNA, is an important research and clinical goal as nucleic acids act to encode and regulate the expression of genes. Conventional detection techniques are based on detecting label or marker substances, or they use label-free sensors.
Label-based sensors use, e.g., fluorescence-based assays to localize and quantitate nucleic acid molecules of interest. However, functionalizing oligonucleotides with fluorescent labels is typically a complex and expensive process that often skews physical and chemical properties, in turn affecting quantitative readout. Label-free sensors circumvent the need for fluorescence modifications, and they are based on, e.g., detecting plasmon resonance, electrochemical conductance or mechanical resonance. However, these techniques may have disadvantages in terms of limited sensitivity or specificity, e.g., due to limited kinetics and thermodynamics of direct hybridization at stringent conditions.
A promising label-free sensor comprises an optical whispering gallery mode (WGM) resonator. See, Vollmer, F. et al., Nanophotonics 1, 267-291 (2012); Fan, X. D. et al., Anal. Chim. Acta 620, 8-26 (2008); Qavi, A. J. et al., Anal. Bioanal. Chem. 394, 121-135 (2009); Hunt, H. K. et al., Nanoscale 2, 1544-1559 (2010); Vollmer, F. et al., Nat. Methods 5, 591-596 (2008); Yoshie, T. et al., Sensors 11, 1972-1991 (2011).
WGM resonators are micron scale optical cavities, such as glass microspheres, capable of confining light by total internal reflection in a small modal volume and only at specific resonance frequencies (resonance wavelengths). These tiny optical resonators exhibit ultra-narrow linewidth, associated with very high quality Q factor, and are extremely sensitive to the binding of biomolecules to the microcavity resonator surface. The changes in permittivity upon binding of analyte result in a shift of the resonance frequency. The high Q factor enables the precise monitoring of small resonance frequency shifts, a method known as the reactive biosensing principle. Vollmer, F. et al., Nanophotonics 1, 267-291 (2012). With the conventional WGM resonator, the resonance frequency is decreased depending on an increasing mass load in response to a specific binding reaction with a target molecule under investigation. The target molecule can be detected by monitoring the negative frequency shift. Optical WGM sensors are emerging as one of the most versatile and sensitive label-free detecting techniques, providing various mechanisms for sensing, sizing, trapping, and manipulation down to the nanoscale. Vollmer, F. et al., Nanophotonics 1, 267-291 (2012); Lin, S. Y. et al., Lab Chip 11, 4047-4051 (2011); Lu, T. et al., Proc. Natl. Acad. Sci. U.S.A. 108, 5976-5979 (2011); Lopez-Yglesias, X. et al., J. Appl. Phys, 111 (2012).
Advantageously, WGM sensors are simple to fabricate, can be functionalized as well as multiplexed, and are made from inexpensive optical fibers. See, Fan, X. D. et al., Anal. Chim. Acta 620, 8-26 (2008); Hunt, H. K. et al., Nanoscale 2, 1544-1559 (2010); Yoshie, T. et al., Sensors 11, 1972-1991 (2011); Vollmer, F. et al., Proc. Natl. Acad. Sci. U.S.A. 105, 20701-20704 (2008); and Qavi, A. J. et al., Angew. Chem.-Int. Edit. 49, 4608-4611 (2010). However, as a general disadvantage, sequence-specific detection by direct DNA hybridization on WGM sensor devices, faces three important challenges: limited sensitivity, specificity, and reusability. See, Fan, X. D. et al., Anal. Chim. Acta 620, 8-26 (2008); Qavi, A. J. et al., Angew. Chem.-Int. Edit. 49, 4608-4611 (2010); Nakatani, K. et al., Chembiochem 5, 1623-1633 (2004); Vollmer, F. et al., Biophys. J. 85, 1974-1979 (2003); Qavi, A. J. et al., Anal. Chem. 83, 6827-6833 (2011); and Suter, J. D. et al. et al., Biosens. Bioelectron. 23, 1003-1009 (2008).
First, years of work on advancing the device physics and engineering of WGM sensors has improved the ultimate physical detection limits of WGM transducers, yet the limits for DNA detection by hybridization has plateaued. Novel molecular approaches are needed to overcome those limitations, mostly set by the inherent kinetics and thermodynamics of the process of molecular recognition through direct hybridization at the sensor surface.
Second, label-free sensors based on hybridization struggle with single base specificity and SNP detection, due to the thermodynamic favorability of hybridization of non-cognate analytes with highly similar sequence to that of the analyte. Although specificity for any particular nucleic acid analyte/probe pair can be optimized by solution salinity and temperature, this process is time consuming and imperfect, and not conducive to significant multiplexing. Similarly, the suppression of nonspecific interactions is essential to multiplexed detection. Thus, non-cognate sequences that differ slightly in sequence may bind non-specifically to the functionalized surface of conventional sensors, generating false positive signals and preventing proper detection.
Finally, conventional label-free nucleic acid detection technologies based on hybridization suffer from the limitation that a different functionalized device is needed to detect each different sequence. Furthermore, each device can generally be only used once; dehybridizing oligonucleotides requires harsh buffer conditions, high temperature, or practically takes too long. See, Lee, M. et al., Anal. Biochem. 282, 142-146 (2000). Thus, different sensors must be constructed to detect different nucleic acid sequences.
DNA strand displacement techniques have recently emerged as a novel family of approaches to enzyme-free homogenous detection assays. See, Zhang, D. Y. et al., Nat. Chem. 4, 208-214 (2012); Yin, P. et al., Nature 451, 318-U314 (2008); Li, B. L. et al., Nucleic Acids Res. 39 (2011); and Zhang, D. Y. et al., Science 318, 1121-1125 (2007). Strand displacement circuits, for example, have been demonstrated to implement nucleic acid “catalysis” in which a nucleic acid sequence of interest effects the release of up to 100 nucleic acid molecules from metastable precursors; cascading such catalytic systems has shown overall turnover of about 1000. Recently, strand displacement has been engineered to allow ultraspecific hybridization assays with specificity approaching the theoretical limit based on thermodynamics. Although strand displacement techniques have significantly improved the specificity and sensitivity of homogeneous detection assays, the readout for this technology has previously been constrained to gel electrophoresis or fluorescence readout, neither of which is easily applicable to point-of-care or clinical diagnostics. See, Zhang, D. Y. et al., Nat. Chem. 4, 208-214 (2012); Zhang, D. Y. et al., Science 318, 1121-1125 (2007); Zhang, D. Y. et al., Nat. Chem. 3, 103-113 (2011); and Zhang, D. Y. et al., J. Am. Chem. Soc. 131, 17303-17314 (2009).